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J. Biol. Chem., Vol. 283, Issue 38, 26047-26058, September 19, 2008

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Differential Roles of Phosphatidylserine, PtdIns(4,5)P₂, and PtdIns(3,4,5)P₃ in Plasma Membrane Targeting of C2 Domains

MOLECULAR DYNAMICS SIMULATION, MEMBRANE BINDING, AND CELL TRANSLOCATION STUDIES OF THE PKC C2 Domain^*

Debasis Manna¹, Nitin Bhardwaj¹², Mohsin S. Vora^¶, Robert V. Stahelin^||, Hui Lu³, and Wonhwa Cho⁴

From the Departments of Bioengineering and Chemistry, University of Illinois, Chicago, Illinois 60607 and the ^¶Department of Biochemistry and Molecular Biology, Indiana University School of Medicine and the ^||Department of Chemistry and Biochemistry and The Walther Center for Cancer Research, University of Notre Dame, South Bend, Indiana 46617

Many cytosolic proteins are recruited to the plasma membrane (PM) during cell signaling and other cellular processes. Recent reports have indicated that phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) that are present in the PM play important roles for their specific PM recruitment. To systematically analyze how these lipids mediate PM targeting of cellular proteins, we performed biophysical, computational, and cell studies of the Ca²⁺-dependent C2 domain of protein kinase C (PKC) that is known to bind PS and phosphoinositides. In vitro membrane binding measurements by surface plasmon resonance analysis show that PKC-C2 nonspecifically binds phosphoinositides, including PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃, but that PS and Ca²⁺ binding is prerequisite for productive phosphoinositide binding. PtdIns(4,5)P₂ or PtdIns(3,4,5)P₃ augments the Ca²⁺- and PS-dependent membrane binding of PKC-C2 by slowing its membrane dissociation. Molecular dynamics simulations also support that Ca²⁺-dependent PS binding is essential for membrane interactions of PKC-C2. PtdIns(4,5)P₂ alone cannot drive the membrane attachment of the domain but further stabilizes the Ca²⁺- and PS-dependent membrane binding. When the fluorescence protein-tagged PKC-C2 was expressed in NIH-3T3 cells, mutations of phosphoinositide-binding residues or depletion of PtdIns(4,5)P₂ and/or PtdIns(3,4,5)P₃ from PM did not significantly affect the PM association of the domain but accelerated its dissociation from PM. Also, local synthesis of PtdIns(4,5)P₂ or PtdIns(3,4,5)P₃ at the PM slowed membrane dissociation of PKC-C2. Collectively, these studies show that PtdIns(4,5)P₂ and PtdIns(3,4,5)P₃ augment the Ca²⁺- and PS-dependent membrane binding of PKC-C2 by elongating the membrane residence of the domain but cannot drive the PM recruitment of PKC-C2. These studies also suggest that effective PM recruitment of many cellular proteins may require synergistic actions of PS and phosphoinositides.

Received for publication, April 4, 2008 , and in revised form, June 23, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants P01AI060915 (to H. L.) and GM52598, GM68849, and GM76581 (to W. C.). This work was also supported by an Indiana University Biomedical Research Grant, American Heart Association Grant SDG 0735350N, and American Cancer Society Grant IRG-84-002-22) (to R. V. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors equally contributed to this work.

² Supported by a fellowship from FMC Technologies, Inc.

³ To whom correspondence may be addressed: Bioinformatics Program, Dept. of Bioengineering (M/C 563), University of Illinois, Chicago, IL 60607. Tel.: 312-413-2021; Fax: 312-413-2018; E-mail: huilu{at}uic.edu. ⁴ To whom correspondence may be addressed: Dept. of Chemistry (M/C 111), University of Illinois, 845 West Taylor St., Chicago, IL 60607-7061. Tel.: 312-996-4883; Fax: 312-996-0431; E-mail: wcho{at}uic.edu.

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